Autoensamblaje de película delgada del BCP2-TPP2ol PDMS-b-PS-TPP2ol con CuBr 2 , producida

The reaction mix was undertaken as follows: 1 pi o f genomic DNA (20 ng), Buffer 10X (to a final concentration of IX), 0.4 pM of each primer, 1.5 mM of MgCb, 200 pM o f each dNTP and distilled water to 50 pi. The PCR program employed was as follows: initial incubation of 96°C for 1 min 30 secs and 29 cycles at 96°C for 30 secs, at 55°C for 50 secs, 68°C for 90 secs and a final extension of 68°C for 10 mins. Afterwards, the products obtained were checked in a non-denaturing agarose gel.

Chapter UI. Straw construction and URA3 espression levels

3.3 Results

3.3.1 Strain construction

3.3.1.1 Replacement of the endogenous mutated URA3 by KanMX

Replacement of the endogenous URA3-52 gene was undertaken by PCR amplification and subsequent transformation. The endogenous URA3-52 is 830 bp and located on chromosome V. For the replacement of this fragment, three PCR reactions were carried out separately, two to amplify the flanking regions of endogenous URA3-

52 and the third one to amplify the gene {KanMX) which would replace the

endogenous URA3-52 gene. Afterwards, the three PCR products were fused by two PCR reactions. Finally, the obtained PCR product {KanMX with URA-52 flanking sequence) was transformed into the original strain using the lithium acetate method (described by Gietz et al., 1992). The endogenous URA3 was replaced by KanMX via homologous recombination with the flanking sequence of URA3-52. The procedure is described below in more detail.

KanM X amplification

The amplification of KanMX, located in the pUG6 (Euroscarf) plasmid was undertaken using PCR (Gueldener et a l, 2002; Guldener et al., 1996). The primers employed in this reaction (Appendix I: URA3-KanMX-V and URA3-KanMX-R) had two regions, one region was homologous to the endogenous URA3 flanking sequence (-40 bp) and the other is homologous to the \oxP-KanMX-\oxP (figure 3.3).

pUC6 KanMX KanMX F KanMX R LoxP PC R product C 775 bp KanMX LoxP LoxP

Figure 3.3 Amplification o f loxP-KanMX-loxP located on plasmidpUG6. The black region of the primer is homologous to the \oxP-KanMX-\oxV sequence in the plasmid and the blue region is homologous to the flanking region of the endogenous URA3 gene.

Amplification of the endogenous URA3-52 flanking sequences

Both flanking sequences of the endogenous URA3-52 were amplified using genomic DNA as a template (figure 3.4). After PCR. the generated products were 394 bp (flanking sequence upstream of URA3-52) and 475bp (flanking sequence downstream of URA3-52). The primers used were termed URA3-1 and URA3-2 for the 394 bp PCR product, whereas, for the 475 bp PCR product the primers used were

URA3-3 and URA3-4 (see Appendix I for primers details). URA3-52 URA3-3 URA3- 1 URA3-4 URA3-2 PCR product A PCR product B

Figure 3.4 Amplification o f the endogenous URA3-52 flanking sequence at the

genomic DNA. The primers used were homologous to the flanking sequence of URA3- 52 in the genomic DNA.

Fusion of the PCR products

The PCR products obtained were purified with a PCR purification kit (Qiagen) and fused by two PCR (figure 3.5). In the first fusion PCR, the 394 bp fragment and loxP-AhwMY-loxP fragments were fused and in the second fusion PCR, the PCR product obtained from the first fusion PCR was fused with the 475 bp long fragments. Both reactions were undertaken using the expand high fidelity PCR system (Roche diagnostic). Before the fusion PCR. the three purified PCR products were diluted 2000x and mixed. Thus, for the first fusion PCR 394 bp and loxP-Ah«MY-loxP fragments were diluted 2000x and mixed together, whereas, for the second fusion PCR the obtained PCR product in the first fusion PCR and the 475 bp fragment were diluted and mixed together. Afterwards, for each fusion PCR, 2 pi of the mixed DNA were used. The programs employed were as follows: initial incubation at 94°C for 4 mins and then 4 cycles of 94°C for 15 secs, 52°C for 3 mins and 68°C for 4 mins with a final extension of cycle at 68°C for 10 mins. This PCR program was followed by a second PCR program with initial incubation at 94°C for 1 min and them 29 cycles of 94°C for 15 secs, 51°C for 30 secs and 68°C for 2 mins and a final extension of 68°C for 10 mins.

hapter III. Strain construction and UR.43 espression levels

3.3.1.2 Transformation and DNA extraction

The fused PCR product was transformed into the original strain by the lithium acetate method (described by Gietz et al., 1992) where the transformed PCR product replaced the endogenous URA3-52 by homologous recombination with the flanking sequence of the endogenous URA3-52, replacing the endogenous URA3-52 with

KanMX. The DNA of the transformed colonies was extracted using glass beads as

(described by Hoffman and Winston, 1987). Transformed colonies were analysed by PCR with £/&43-outside-F and LTLfi-outside-R primers (Appendix I) to select for positive colonies. Afterwards, the obtained PCR products were analysed via an agarose gel (figure 3.6). The new strains obtained were named FEP 178ura3-52A and

i* £1-1 i UU i Uw#1wj " J iij = I st Fusion PCR: URA3-1 775 bp PCR product C URA3-KanMX-R 2 nd F u sion P C R : U R A 3 -I 1169 bp PC R product A + C

I

URA3-52 LoxP 1634 bp PC R product A + B + C KanMX LoxP

I

URA3-4 475 bp PC R product B URA3-52 flanking region

Transform ation and integration in the genom e by recom bination

Figure 3.5 Fusion o f three PCR products. The first fusion PCR was undertaken with URA3-1 and URA3-KanMX-R primers and as templates KanMX amplification product and 394 bp length PCR product. The obtained PCR product (PCR product A+C) was 1169 bp. The second fusion PCR was carried out with URA3-1 and URA3-

4 primers. As templates the first fusion PCR product (PCR product A+C) and 475 bp length URA3 flanking sequence PCR product were used. The final product was 1634 bp (PCR product A+B+C) and it was transformed into yeast strains so it could integrate in the genome by recombination.

> 1 Kb 2 - 1Kb s '

1.6Kb _{1.6 kb}

Figure 3.6 DNA from the transformant colonies with endogenous URA3-52 replaced

with KanMX. The transformants obtained from the FEP 100-10 strain are represented

as 1 and the transformant obtained from the FEP178 strain is represented as 2 following by the genomic negative control.

3.3.1.3 Replacement of the endogenous SIR2 gene with LEU2

The SIR2 gene is located at chromosome IV and is 1689 bp long. The replacement was undertaken with an amplification of the LEU2 gene (1128 bp), [from the pUG73 plasmid (Euroscarf) with LEU2R and LEU2V primers (see Appendix I)]. The primers used for LEU2 amplification contain two regions, one of them homologous to the SIR2 flanking sequence (5'side of the primer) and the other region is homologous to the plasmidic LEU2 (3'side of the primer) (figure 3.7). The PCR was undertaken as previously described in this chapter (section 3.3.1.1).

The transformation followed by DNA extraction was then conducted as described in this chapter, (section 3.3.1.2) and the positive colonies were checked by PCR with SIR2F2 and SIR2R(2) primers (Appendix I). The obtained PCR amplicon was analysed in an agarose gel (figure 3.8) and the generated strains were called FEP1 !Ssir2 * and FEP100-10sir2 *

Chapter 111. Strain construction unci l-RAS expression Use o,

pUG73

L E U 2F

LEU2

L E U 2R

Figure 3.7 Amplification ofLEU2 located in the pUG73 plasmid. The black region of the primer is homologous to the LEU2 in the plasmid and the purple region is homologous to the flanking region of endogenous SIR2 in the genome.

FEP 178Sir2* FEP 100-10Sir2*

Figure 3.8 DNA from transformant colonies with endogenous SIR2 replaced by LEU2

gene. The positive control with the FE?\18ura3-52::KanMX genomic DNA followed

by the transformant colonies from EE?\18ura3-52: :KanMX and from FEP 100-10

ura3-52::KanMX strains. The positive clones are illustrated in bold.

3.3.2 Subtelomeric URA3 expression in the constructed strains

The expression of the subtelomeric URA3 gene was measured to determine whether there were differences in silencing levels between different native chromosomes ends, and expression was compared to that of the endogenous URA3 gene. URA3 gene expression was also measured in the SIR2 deleted strains to examine whether the deletion of SIR2 could affect the silencing and the expression of URA3. The expression of URA3 was determined by measuring the fraction of cells that were able to grow on plates containing 5-Fluoroorotic acid (5-FOA) versus the colonies able to grow on plates without 5-FOA. As mentioned before, 5-FOA is only toxic when the URA3 gene product is present in the cell. Cells expressing URA3 are unable to grow on 5-FOA and cells that do not express URA3 become 5-FOA resistant and therefore can form colonies.

The results are summarised in figure 3.9 and in Appendix II. The figure shows the percentage of surviving colonies in each medium. When URA3 is inserted in the XI-L (FEP100-10ura3-52A), there is no difference in the number of surviving colonies in the presence or absence of uracil in the different growth media, 54.8±8 % surviving colonies on plates with uracil and 49.1±18.9 % on plates without uracil. When URA3 is inserted at III-R (FEP178) the number of surviving colonies is reduced by 20-fold in media with uracil (5.2±6.5 %) and 220-fold in media without uracil (2.5±0.57 %). When the SIR2 gene is deleted, the survival of both strains was below 0.01 % irrespective of uracil status. As a control, the expression of the endogenous

URA3 was also analysed. The results were as expected; when URA3 is at its natural

location it is only expressed in the absence of uracil or starvation conditions (0.2±0.3 %) but not in the presence of uracil (94.6±57 %).

This experiment showed that the level of URA3 expression differs when it is located at different chromosome ends; when the URA3 gene is inserted into the III-R (FEP 178) chromosome, the level of repression is very low. In contrast, when URA3 is inserted at the XI-L (FEP 100-10) chromosome end, the level of repression is much higher. When the SIR2 gene is deleted the URA3 is expressed at the same level at both chromosome ends. The regulation of the expression at the different chromosome ends also differs from that of the endogenous URA3 gene. This confirms that the different chromosome ends have a distinct grade of silencing as previously (reported in Pryde and Louis, 1999). They analysed the expression of inserted URA3 at different subtelomeric regions and at different chromosome ends.

The results shown in this thesis additionally suggest that repression at the subtelomeres is due to the spreading of silencing since when silencing is disrupted via deletion of the SIR2 gene, the URA3 gene at both subtelomeric regions is expressed.

Chapter 111. Strain construction and URA3 espression levels

■ URA- ■ URA+

RE(XIL) REsir2A(XIL) NRE(1IIR) NR£sir2A(IlIR) Endogenous URA3

Figure 3.9 Expression o f the URA3 gene for all strains employed. Colonies grown in minimal medium with uracil are indicated in orange. Colonies grown in minimal medium without uracil are represented in green. The strains used were NRE (URA3 inserted at III-R), NRE.sz>2A (URA3 inserted at III-R and deletion of SIR2), RE

{URA3 inserted at XI-L), REs/r2A (URA3 inserted at XI-L and deletion of SIR2).

In order to simplify nomenclature, the strain where URA3 is inserted in the III-R was termed the non repressive end (NRE) and the strain where URA3 is inserted in the XI-L was termed the repressive end (RE) throughout this thesis. When SIR2 was deleted the strains where called NRE.v/r2A and RE.S7>2A.

3.4 Discussion

3.4.1 Expression of the subtelomeric URA3 gene at different chromosome ends

In document CENTRO DE INVESTIGACIÓN EN QUÍMICA APLICADA Programa de Doctorado en tecnología de polímeros TESIS (página 158-162)